Surface treatment method for copper or copper alloy, surface treatment liquid for sterilizing copper or copper alloy, and sterilization method using copper or copper alloy treated by said method

ABSTRACT

The present invention provides a surface treatment method that improves antimicrobial activity of copper or a copper alloy and enhances immediate effects of antimicrobial actions on the surface of the copper or the copper alloy. A surface treatment method for copper or a copper alloy according to the present invention comprises preparing a reducing agent solution containing a biological reducing substance, and treating the surface of the copper or the copper alloy with the reducing agent solution. The present invention also provides a surface treatment liquid for sterilizing copper or a copper alloy, in which the surface treatment liquid contains a biological reducing substance. The present invention also provides a sterilization method that comprises bringing copper or a copper alloy treated by the surface treatment method into contact with a surface of an object to sterilize the surface of the object.

TECHNICAL FIELD

The present invention relates to a surface treatment method for copperor a copper alloy, a surface treatment liquid for sterilizing copper ora copper alloy, and a sterilization method using copper or a copperalloy treated by the method.

BACKGROUND ART

In recent years, the spread of antimicrobial-resistant bacteria andviruses having resistance to antimicrobials and antivirals has beenregarded as a serious problem. A collaborative study between the UKGovernment and the Wellcome Trust estimates that 10 million peopleacross the world will die by 2050 because of antimicrobial-resistantbacteria (Review on Antimicrobial Resistance. Antimicrobial Resistance:Tackling a Crisis for the Health and Wealth of Nations. 2014). Thisnumber exceeds the number of cancer fatalities each year, and theassociated economic loss is estimated at about 100 trillion dollars.WHO's global action plan against infection caused byantimicrobial-resistant bacteria (Global Action Plan on AntimicrobialResistance. World Health Organization. 2015) requires urgent action todeal with a flurry of development of antimicrobial resistance. Thedocument also describes how to reduce the incidence of infection, how tooptimize the use of antimicrobials, and the importance in complying withthe optimal use.

In the midst of growing threats caused by antimicrobial-resistantbacteria and viruses, metallic copper (hereinafter also simply referredto as “copper”) and alloys thereof are attracting attention as newantimicrobial and antiviral materials. This is because materials'surfaces touched by human hands are confirmed to play an important rolein preventing infection. A material surface is not only a source ofinfection due to bacteria attached to the surface from human hands butalso a cause of horizontal transfer of antimicrobial-resistance genesdue to coexistence of several types of bacteria. Therefore, not only newantimicrobial-resistant bacteria but also multiantimicrobial-resistantbacteria may be generated. In order to prevent the generation of suchbacteria, continuous disinfection of materials surfaces is necessary,and copper and copper alloys have attracted attention as such materials.It is reported that a surface of copper and a copper alloy exhibitsstrong bactericidal actions which destroy bacteria attached to thesurface and degrade DNA of the bacteria in about several tens of minutesto several hours. Due to the gene degradation, it is determined thatcopper and copper alloys do not cause horizontal transfer that generatesnew antimicrobial-resistant bacteria and that copper and copper alloysare effective against many types of pathogens such as viruses and fungias well as bacteria.

Accordingly, introduction of copper and copper alloy products arepromoted in medical institutions, nursing homes, and places where manypeople gather (such as nurseries and public transportation vehicles).For example, in a demonstration experiment in a hospital, Non PatentLiterature 1 reports an 83% reduction in bioburden (the number of viablemicrobes in an environment), and Non Patent Literature 2 reports a 58%reduction in incidence of healthcare-acquired infections (HAI).

CITATION LIST Non Patent Literature

-   Non Patent Literature 1: M. G. Schmidt, et al. Journal of Clinical    Microbiology 50 (2012) 2217-2223-   Non Patent Literature 2: C. D. Salgado, et al. Infection Control and    Hospital Epidemiology 34 (2013) 479-486

SUMMARY OF INVENTION Technical Problem

Impressively, as described in the aforementioned reported case, eventhough an 80% or more reduction in bioburden is determined, bioburden isnot reduced to zero, and the incidence of HAI is reduced by about half.One possible reason is that it takes time to manifest antimicrobialactions on a surface of copper and a copper alloy. If the antimicrobialactions on the surface of the copper and the copper alloy are manifestedin few minutes, or tens of seconds, or few seconds but not in few hours,it may be possible to diminish the risks of infectious diseases and theaforementioned horizontal transfer of antimicrobial-resistance genes.

The present invention has been made in light of the circumstances, andan object of the present invention is to provide a method that improvesantimicrobial activity of copper or a copper alloy and enhancesimmediate effects of antimicrobial actions on a surface.

Solution to Problem

In order to achieve the object, the present inventors have paidattention to reactive oxygen species, one of the mechanisms formanifesting antimicrobial actions of copper and a copper alloy.

Reactive oxygen species are considered to be generated in the livingbody by the Fenton reaction. The Fenton reaction is a reaction in whichiron (II) ions and hydrogen peroxide (H₂O₂) are combined in an aqueoussolution to generate hydroxyl radicals (—OH). It is known that a similarreaction occurs with copper (I) ions. A mechanism for generatinghydrogen peroxide on a surface of copper and a copper alloy has yet tobe revealed. However, hitherto, 0.5 to 2.0 mg/L of hydrogen peroxide isgenerated in 10 to 20 minutes after a contact of a LB-medium with seededEscherichia coli to a surface of pure copper (Shigetoshi Kobuchi et al.have reported in Zairyo-to-Kankyo/Corrosion Engineering of Japan, 52(2003), pp. 428-435). In the same document, a slight amount of hydrogenperoxide is generated by a contact of a similar LB-medium to a surfaceof pure silver, but not in case of gold. Furthermore, they have reportedno generation of hydrogen peroxide by a contact of a simple LB-medium toa surface of pure copper.

It is also considered that copper (II) ions eluted from a surface ofcopper and a copper alloy have something to do with bactericidal actionson the surface of copper and the copper alloy (for example, G. Grass etal. Appln. Environ Microbiol. 77 (2011) 1541-1547). Specifically, thefollowing mechanisms have been inferred which potentially actindependently or interact with each other; A) copper (II) ions elutedfrom a surface of copper or a copper alloy damage bacterial cells incontact with the surface; B) the copper (II) ions (and otherstress-inducing events) destroy cellular membranes, causing loss ofmembrane potential and loss of cytoplasmic components; C) the copper(II) ions induce generation of hydroxyl radicals and reactive oxygenspecies, causing further damage to the cells; D) the bacteria damaged bythe copper (II) ions decays, resulting in degradation of genomic DNA andplasmid DNA. There are still unclear points in these mechanisms ofaction. For example, microbes have important metabolic enzymes in cellwalls, which are easily interact with extracellular copper (II) ions.

As described above, on a surface of copper and a copper alloy,bactericidal and antimicrobial actions are manifested by additive andsynergistic effects of both elution of copper (II) ions and generationof hydrogen peroxide. Such findings give the present inventors an ideathat promoting generation of hydrogen peroxide and elution of copper(II) ions on a surface of copper and a copper alloy enables a fastertime to manifest bactericidal actions on the surface. As a result ofintensive studies on substances and methods that exhibit such functions,the present inventors have found that treatment of a surface of copperor a copper alloy with a reducing substance in vivo (hereinafterreferred to as “biological reducing substance”) effectively generateshydrogen peroxide on the surface and that reactions between hydrogenperoxide and copper (I) ions on the surface of the copper or the copperalloy promotes generation of reactive oxygen species, which enhancesmanifesting efficiency of antimicrobial actions.

The findings of the present inventors are based on the thought that theaforementioned relation between reactive oxygen species, hydrogenperoxide, copper (I) ions, and copper (II) ions is represented by thefollowing chemical equations (1) to (3) associated with actions of areducing agent:2Cu²⁺+2RSH→2Cu⁺+RSSR+2H⁺  (1)2Cu⁺+2H⁺+O₂→2Cu²⁺+H₂O₂  (2)Cu⁺+H₂O₂→Cu²⁺+OH⁻+—OH  (3)

(In Formula (1), RSH is a reducing agent, and RSSR is an oxidant of thereducing agent. Although thiol RSH is described as the reducing agent,note that thiol RSH is a typical example of the biological reducingsubstance. There is no intention to exclude reducing agents other thanthiol-based substances).

Here, with regard to the bactericidal actions caused by Formulae (1) to(3), reactive oxygen species is principally generated even in an aqueoussolution if copper ions and an appropriate reducing agent are involved.However, in order to generate a sufficient amount of reactive oxygenspecies for sterilization, copper ions at a fairly-high concentrationare required. In addition, since reactive oxygen species has a shortlifetime and moves in a very short distance, reactive oxygen species iseffective only around the place where it is generated. Therefore, thefact that a region near a surface of an article made of copper or acopper alloy contains eluted ions at a higher concentration than that ina copper ion-containing liquid, or the fact that the surface of thearticle includes copper oxide (Cu₂O) that proceeds the aforementionedreactions on the surface, as described later, enables efficientsterilization.

Based on these new findings, the present inventors have made furtherstudies and resulted in the present invention.

In an aspect of the present invention, there is provided a surfacetreatment method for copper or a copper alloy, in which the methodcomprises preparing a reducing agent solution containing a biologicalreducing substance, and treating a surface of copper or a copper alloywith the reducing agent solution.

Here, the copper or the copper alloy may be a wrought copper and copperalloy product.

The copper or the copper alloy may be a fiber, a particle, or foil ofcopper or a copper alloy contained in a porous body.

The porous body may be a woven fabric, a non-woven fabric, or a sponge.

Further, the treating may be performed at a relative humidity of 70% RHor less.

The copper or the copper alloy may be a Cu—Zn alloy, a Cu—Ni—Zn alloy, aCu—Sn—Ni—Zn alloy, or a Cu—Si—Pb—P—Zn alloy.

The biological reducing substance may be at least one of reducedglutathione, N-acetylcysteine, sodium ascorbate, sodium sulfite, andcysteine.

The biological reducing substance may be reduced glutathione, and thereduced glutathione in the reducing agent solution may have aconcentration of be 0.5 to 2.0 mM.

The copper or the copper alloy may include an oxide layer, and the oxidelayer may contain 80.0% or more of Cu₂O and 20.0% or less of CuO in anarea from the surface of the oxide layer to 1 μm measured by XPS.

In another aspect of the present invention, there is provided a surfacetreatment liquid for sterilizing copper or a copper alloy, in which thesurface treatment liquid contains a biological reducing substance.

Here, the biological reducing substance may be at least one of reducedglutathione, N-acetylcysteine, sodium ascorbate, sodium sulfite, andcysteine.

In another aspect of the present invention, there is provided asterilization method that comprises bringing copper or a copper alloytreated by the surface treatment method into contact with a surface ofan object to sterilize the surface of the object.

Here, the copper or the copper alloy may be a fiber, a particle, or foilof copper or a copper alloy contained in a porous body.

The porous body may be a woven fabric, a non-woven fabric, or a sponge.

Advantageous Effects of Invention

According to the present invention, there is provided a surfacetreatment method that improves antimicrobial activity of copper or acopper alloy and enhances immediate effects of antimicrobial actions onthe surface of the copper or the copper alloy.

Further, according to the present invention, there is provided a surfacetreatment liquid for sterilizing copper or a copper alloy, in which thesurface treatment liquid contains a biological reducing substance.

Furthermore, according to the present invention, there is provided asterilization method using copper or a copper alloy treated by theaforementioned method.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows results of hydrogen peroxide (H₂O₂) generation amountsmeasured in Example 1 by immersing sample pieces of Cu, CBRI, CBRA, andC6932 in reducing agent solutions containing different biologicalreducing substances.

FIG. 2 shows results of hydrogen peroxide (H₂O₂) generation amountsmeasured in Example 1 by immersing a Cu sample piece in reducing agentsolutions containing different biological reducing substances.

FIG. 3 shows results of hydrogen peroxide (H₂O₂) generation amountsmeasured in Example 2 by immersing sample pieces of Cu, C2680, CBRI, andCBRA in reducing agent solutions containing GSH having differentconcentrations.

FIG. 4 shows results of hydrogen peroxide (H₂O₂) generation amountsmeasured in Example 3 by immersing sample pieces of Cu, C2680, C6932,CBRI, and CBRA in reducing agent solutions containing different solventsto which GSH was added at a concentration of 1.0 mM.

FIG. 5 shows results of hydrogen peroxide (H₂O₂) generation amountsmeasured in Example 4 by immersing sample pieces of Cu, C2680, C6932,CBRI, CBRA, antimicrobial SS, and Ag in reducing agent solutions towhich GSH was added at a concentration of 1.0 mM.

FIGS. 6(a) and 6(b) are schematic views showing an outline of a simpletest for antimicrobial activity using Escherichia coli (E. coli) inExample 5.

FIG. 7 shows results of the simple test for antimicrobial activity usingE. coli in Example 5.

FIG. 8 shows results of the simple test for antimicrobial activity usingE. coli in Example 5.

FIG. 9 shows results of a simple test for antimicrobial activity usingE. coli in Example 6.

FIG. 10 shows results of a test for antimicrobial activity using E. coliin Example 7.

FIG. 11 shows results of a test for antimicrobial activity usingStaphylococcus aureus (S. aureus) in Example 8.

FIGS. 12(a) and 12(b) show results of a test for antimicrobial activityusing E. coli and S. aureus in Example 9.

DESCRIPTION OF EMBODIMENTS

Hereinafter, embodiments of the present invention will be described.

A surface treatment method for copper or a copper alloy according to anembodiment of the present invention comprises preparing a reducing agentsolution containing a biological reducing substance, and treating asurface of copper or a copper alloy with the reducing agent solution.

In the surface treatment method according to this embodiment, first, areducing agent solution containing a biological reducing substance isprepared.

The biological reducing substance is not particularly limited as long asit functions as a reducing agent (for example, RSH) in the chemicalequation (1). Specific examples of the biological reducing substanceinclude, but are not limited to, reduced glutathione (GSH),N-acetylcysteine (NAC), sodium ascorbate (AA-Na), sodium sulfite(Na₂SO₃), and cysteine (Cys).

A solvent for preparing the reducing agent solution is not particularlylimited as long as it dissolves or suspends the biological reducingsubstance. Specific examples of the solvent include water (H₂O) andorganic solvents but are not limited thereto. Note that the reducingagent solution may be a homogeneous solution containing a biologicalreducing substance completely dissolved in a solvent or may be aheterogeneous solution containing a biological reducing substancedispersed in a solvent.

A concentration of the biological reducing substance in the reducingagent solution is appropriately adjusted according to, for example,types of the biological reducing substance and solvent, and types of thecopper or the copper alloy to be treated. For example, a reducing agentsolution is prepared by adding a predetermined biological reducingsubstance, to a predetermined solvent, within a range of 0.01 to 10.0mM, 0.05 to 7.5 mM, 0.1 to 5.0 mM, 0.5 to 2.0 mM, or 0.75 to 1.5 mM.Note that any additives may be added in the reducing agent solution asneeded.

In the surface treatment method according to this embodiment, next, thesurface of the copper or the copper alloy is treated with the reducingagent solution.

Examples of the copper used in this embodiment include, but are notlimited to, copper having purity of 95% by mass or more, more preferably99.90% by mass or more, such as oxygen-free copper (JIS H3100, alloynumber C1020), tough pitch copper (JIS H3100, alloy number C1100), andphosphorous-deoxidized copper (JIS H3100, alloy numbers C1201 and C1220)specified in JIS H0500 and JIS H3100, and electrolytic copper foil.

In this specification, copper alloys represent alloys containing 50% bymass or more of copper. Examples of the copper alloys used in thisembodiment include, but are not limited to, copper-zinc alloys (brass),copper-nickel alloys (cupronickel), copper-nickel-zinc alloys (nickelsilver), copper-tin alloys (bronze), and copper-tin-phosphorus alloys(phosphor bronze).

In addition, as the copper alloys, for example, it is possible to usebinary alloys having two basic component elements including copper,ternary alloys having three basic component elements including copper,and alloys having four or more basic component elements includingcopper. Examples of the binary alloys include Cu—Zn alloys,specifically, brass (JIS H3100, alloy numbers C2600 and C2680). Examplesof the ternary alloys include Cu—Ni—Zn alloys. The ternary alloyspreferably contain 50.0 to 60.0% by mass of Cu, 5.0 to 15.0% by mass ofNi, with the balance being zinc and inevitable impurities. Examples ofthe copper alloys having four or more basic component elements includeCu—Sn—Ni—Zn alloys and Cu—Si—Pb—P—Zn alloys. Preferably, Cu—Sn—Ni—Znalloys contain 60.0 to 80.0% by mass of Cu, 0.1 to 1.0% by mass of Sn,0.5 to 5.0% by mass of Ni, with the balance being zinc and inevitableimpurities. Preferably, Cu—Si—Pb—P—Zn alloys contain 65.0 to 85.0% bymass of Cu, 1.0 to 5.0% by mass of Si, 0.01 to 1.0% by mass of Pb, 0.01to 0.5% by mass of P, with the balance being zinc and inevitableimpurities.

Taking various conditions into account such as types, sizes, and shapesof copper or a copper alloy to be treated, various known modes such asspraying, coating, and immersion may be appropriately selected as a modefor surface treatment of copper or a copper alloy with a reducing agentsolution.

The surface treatment of copper or a copper alloy with a reducing agentsolution is preferably performed while a surface of copper or a copperalloy to be treated is dry. For example, the surface treatment of copperor a copper alloy with a reducing agent solution is performed in theatmosphere in which a temperature is controlled to 30° C. or less and arelative humidity is controlled to 70% RH or less. However, even thougha temperature is higher than 30° C. or a relative humidity is higherthan 70% RH, it is possible to obtain effects of the present invention.

On a surface of copper or a copper alloy treated with a reducing agentsolution, a biological reducing substance contained in the reducingagent solution reacts with copper (II) ions eluted from the surface, andthis reaction proceeds the chemical equation (1) and generates copper(I) ions. This reaction promotes generation of copper (II) ions andhydrogen peroxide (H₂O₂) by the chemical equation (2) and generation ofcopper (II) ions and hydroxyl radicals (—OH) by the chemical equation(3). Furthermore, the copper (II) ions generated by the chemicalequation (2) or the chemical equation (3) cause another reaction withthe biological reducing substance by the chemical equation (1).Accordingly, compared with a surface of untreated copper or an untreatedcopper alloy, the surface of the copper or the copper alloy is providedwith more copper (II) ions, hydrogen peroxide, and hydroxyl radicals,which enhances immediate effects of antimicrobial actions on the surfaceand increases manifesting efficiency of the antimicrobial actions of thecopper or copper alloy. Such a mechanism of action is inferred with acertain reliability based on the results of the following Examples.

In view of the mechanism of action, in this embodiment, it is consideredthat creating a state with copper (I) present on a surface of copper ora copper alloy is effective in increasing immediate effects ofantimicrobial actions on the surface. This is because copper (II) ions,hydrogen peroxide, and hydroxyl radicals may be generated by thechemical equations (2) and (3) on a surface with copper (I). Generally,copper and copper alloys gradually discolor due to an oxide layer formedon a surface thereof in a normal usage environment. Therefore, as apreliminary test, the present inventors exposed a sample of oxygen-freecopper (C1020) to the normal atmosphere for three months and conductedXPS analysis on the surface of the sample. According to the result, aproportion of Cu₂O and CuO was 83.0% and 17.0%, respectively.

The surface treatment method according to this embodiment is effectivelyapplied to copper or a copper alloy having a polished surface, andapplied to copper and a copper alloy having an oxide layer on thesurface thereof and containing a high proportion of Cu₂O in elementalcompositions determined by XPS analysis. An example of the oxide layerincludes one that contains 80.0% or more of Cu₂O and 20.0% or less ofCuO in an area from the surface of the oxide layer to 1 μm measured byXPS. In addition, the oxide layer is not limited to those formed byexposure to the atmosphere and may be formed, for example, by treating asurface of copper or a copper alloy by any generally applicable method.

In this manner, on a surface of copper or a copper alloy having an oxidelayer containing a large amount of Cu₂O, copper (II) ions are elutedfrom the surface of the metal copper or the copper alloy into theaforementioned reducing agent solution. The elution of the copper (II)ions initiates and proceeds reactions represented by the chemicalequations (1) to (3). In addition, the presence of more copper (I) onthe surface of the copper or the copper alloy results in more copper(II) ions, hydrogen peroxide, and hydroxyl radicals. Accordingly,immediate effects of antimicrobial actions on the surface are furtherenhanced, leading to an increase in manifesting efficiency of theantimicrobial actions of the copper or the copper alloy. Such amechanism of action is inferred with a certain reliability based on theresults of the following Examples.

Furthermore, in a more specific aspect of this embodiment, the copper orthe copper alloy is preferably a wrought copper and copper alloyproduct. In this specification, the wrought copper and copper alloyproduct is a general term for products such as sheets, strips, tubes,rods, and wires of copper and copper alloys produced by hot or coldplastic working such as rolling, extrusion, drawing, and forgingaccording to the definition specified in JIS H0500. Treatment of asurface of such a wrought copper and copper alloy product with thereducing agent solution of this embodiment enables quick and efficientdestruction of bacteria, microbes and the like attached to the surfaceand sterilizes the surface. The wrought copper and copper alloy productto which the surface treatment method according to this embodiment isapplied is not limited to those used independently and may be used incombination with other products. Examples of the wrought copper andcopper alloy product include, but are not limited to, articles,equipment, components, and indoor floors and wall surfaces, in whichcopper or a copper alloy is used in whole or in part. More specifically,the surface treatment method according to this embodiment is effectivelyapplied to members used in environments that require eradication,sterilization, and disinfection, such as kitchen sinks, bathroom walls,medical institutions, nursing homes, and floors and walls ofpharmaceutical and medical equipment manufacturing facilities.

In another specific aspect, the copper or the copper alloy is preferablya fiber, a fine particle, or foil of copper or a copper alloy(hereinafter simply represented by a copper fiber) contained in a wovenfabric, a non-woven fabric, a sponge, or other general flexible porousmaterials (hereinafter represented by a cloth). In this case, thereducing agent solution of this embodiment is applied to the cloth totreat the surface of the copper fiber with the reducing agent solution.The sterilized cloth damp with the reducing agent solution is broughtinto contact with a surface of an object (for example, dry or wipe thesurface) so as to transfer bacteria, microbes, and the like attached tothe surface of the object to the surface of the sterilized cloth andremove those bacteria and the like from the surface. Furthermore,reactive oxygen species and eluted copper ions generated around thecopper fiber quickly and efficiently destroy bacteria, microbes, and thelike. Accordingly, the surface of the object is sterilized.

In this manner, the reducing agent solution according to this embodimentis preferably used for the surface treatment of copper or a copperalloy. In other words, the reducing agent solution according to thisembodiment is preferably used as a surface treatment liquid forsterilizing copper or a copper alloy.

Furthermore, when copper or a copper alloy treated by the surfacetreatment method according to this embodiment is brought into contactwith a surface of an object, it is possible to sterilize the surface ofthe object.

EXAMPLES

Hereinafter, the present invention will be described in more detail withreference to Examples, but the present invention is not limited to thefollowing Examples.

[Copper and Copper Alloy Samples]

The following samples were used as copper and copper alloy samples:

-   -   Oxygen-free copper (alloy number C1020. Also referred to “C1020”        or “Cu”.)    -   Brass (alloy number C2680. Also referred to “C2680”.)    -   Clean Bright (registered trademark) (Mitsubishi Shindoh Co.,        Ltd. Also referred to “CBRI”.) Typical composition: 54.0 Cu-11.0        Ni—Zn (% by mass)    -   Clean Brass (registered trademark) (Mitsubishi Shindoh Co., Ltd.        Also referred to “CBRA”.) Typical composition: 70.0 Cu-0.5        Sn-2.0 Ni—Zn (% by mass)    -   Eco Brass (registered trademark) (Mitsubishi Shindoh Co., Ltd.        Also referred to “C6932”.) Typical composition: 75.5 Cu-3.0        Si-0.09 Pb-0.1 P—Zn (% by mass)

Unless otherwise described in the following Examples, copper and copperalloy samples have no oxide layer on the surface thereof or have anoxide layer (natural oxide layer) to a negligible extent.

Example 1

Reduced glutathione (GSH), N-acetylcysteine (NAC), sodium ascorbate(AA-Na), sodium sulfite (Na₂SO₃), and cysteine (Cys) were used asbiological reducing substances. Reducing agent solutions were preparedby adding each biological reducing substance to water (H₂O) in aconcentration range in which the biological reducing substance isdissolved.

Each sample piece (Cu, CBRI, CBRA, and C6932) having a surface area of0.95 cm² was immersed in each reducing agent solution for 24±1 hours,and then, each hydrogen peroxide (H₂O₂) generation amount was measured.

The H₂O₂ generation amount was calculated based on a difference from thetime of adding H₂O₂ scavenger (catalase) by the xylenol orange (XO)method and shown as a generation amount per unit solution. The followingExamples are carried out in similar conditions.

Results are shown in FIGS. 1 and 2 .

FIG. 1 shows the results of immersion tests on sample pieces of Cu,CBRI, CBRA, and C6932. Reducing agent solutions of 1 mM GSH, 0.01 mMNAC, 0.1 mM AA-Na, and 1.0 mM Na₂SO₃ were used in the immersion testsfor each sample piece, and the results are shown in this order from theleft.

FIG. 2 shows the results of immersion tests on Cu using reducing agentsolutions of NAC (0.01 mM, 0.1 mM, 1.0 mM), AA-Na (0.01 mM, 0.05 mM, 0.1mM), Na₂SO₃ (0.1 mM, 0.5 mM, 1.0 mM, 5.0 mM, 10.0 mM), Cys (0.1 mM), andGSH (1.0 mM).

These results show that, with any reducing substance, H₂O₂ was generatedfrom the samples. Particularly, when using a GSH solution, a certainamount of H₂O₂ was generated in each sample, and H₂O₂ generation amountsin CBRI and CBRA were prominent. Accordingly, GSH was adopted as abiological reducing substance in Example 2 and in subsequent Examples.

In this Example, reducing agent solutions containing one type ofbiological reducing substance were used. For example, when a solution ofCys (0.1 mM) was used, a very small amount of H₂O₂ was generated.However, it should be noted that H₂O₂ generation amount may be increasedin different concentration conditions or by combining with otherbiological reducing substances.

Example 2

In this Example, a relation between a GSH concentration in a reducingagent solution and a H₂O₂ generation amount was studied.

GSH was added to water (H₂O) at a concentration of 0.1 mM, 0.5 mM, and1.0 mM to prepare reducing agent solutions. As in Example 1, immersiontests were performed on Cu, C2680, CBRI, and CBRA, and each H₂O₂generation amount was measured.

For comparison, a similar immersion test was performed using a 500-folddiluted nutrient broth ( 1/500 NB) under conditions based on the testfor antimicrobial activity specified in JIS Z2801.

Results are shown in FIG. 3 .

FIG. 3 shows the results of the immersion tests on sample pieces of Cu,C2680, CBRI, and CBRA. Reducing agent solutions of 1/500 NB, 0.1 mM GSH,0.5 mM GSH, and 1.0 mM GSH were used in the immersion tests for eachsample piece, and the results are shown in this order from the left.

In each sample piece, when a GSH concentration was 0.1 mM, a H₂O₂generation amount was very small. The results also show that a H₂O₂generation amount increases depending on a GSH concentration.Particularly, when a GSH concentration was 1.0 mM, an increase in H₂O₂generation amount was prominent compared with cases where GSHconcentrations were 0.1 mM and 0.5 mM.

Example 3

In this Example, a GSH concentration was fixed at 1.0 mM, and a relationbetween a solvent of a reducing agent solution and a H₂O₂ generationamount was studied.

GSH was added at a concentration of 1.0 mM to 500-fold diluted nutrientbroth ( 1/500 NB), water (H₂O), 0.9% NaCl, and 5% NaCl to preparereducing agent solutions. As in Example 1, immersion tests wereperformed on Cu, C2680, C6932, CBRI, and CBRA, and each H₂O₂ generationamount was measured.

Results are shown in FIG. 4 .

FIG. 4 shows the results of the immersion tests on sample pieces of Cu,C2680, C6932, CBRI, and CBRA. Reducing agent solutions in which GSH wasadded at a concentration of 1.0 mM to 1/500 NB, H₂O, 0.9% NaCl, and 5%NaCl were used in the immersion tests for each sample piece, and theresults are shown in this order from the left.

These results indicate that a solvent contained in a reducing agentsolution is appropriately selected according to, for example, types ofcopper or copper alloys, types of biological reducing substances, andconcentrations of biological reducing substances in the reducing agentsolution.

Example 4

In this Example, a reducing agent solution containing 1.0 mM GSH wasused, and H₂O₂ generation amounts in various copper and copper alloysamples were measured.

As a condition based on the test for antimicrobial activity specified inJIS Z2801, GSH was added at a concentration of 1.0 mM to 5 mL of500-fold diluted nutrient broth ( 1/500 NB), whereby preparing areducing agent solution. Immersion tests were performed in a similarmanner to Example 1 on Cu, C2680, C6932, CBRI, and CBRA and also onantimicrobial stainless steel (antimicrobial SS) and metallic silver(Ag) as samples for comparison. Then, each H₂O₂ generation amount wasmeasured.

For comparison, each sample was subjected to a similar immersion testusing 5 mL of 500-fold diluted nutrient broth ( 1/500 NB) without GSH.

Results are shown in FIG. 5 .

FIG. 5 shows the results of the immersion tests on sample pieces of Cu,C2680, C6932, CBRI, CBRA, antimicrobial SS, and Ag. The bar graph on theleft of each sample piece shows the result of the immersion test using1/500 NB to which GSH was added at a concentration of 1.0 mM, and thebar graph on the right shows the result of the immersion test using1/500 NB without GSH.

With the reducing agent solution of 1/500 NB to which GSH was added,H₂O₂ was generated from each sample. It was also determined that a H₂O₂generation amount differs depending on types of samples.

Example 5

In this Example, effects of GSH, which is a biological reducingsubstance, on antimicrobial activity of copper and copper alloys werestudied by a simple test for antimicrobial activity using E. coli basedon the test for antimicrobial activity specified in JIS Z2801.

FIGS. 6(a) and 6(b) are schematic views showing an outline of the testfor antimicrobial activity performed in this Example.

As shown in FIG. 6(a), first, a copper (Cu) sample piece (15 mm square)was placed on the bottom surface of a glass container. Next, on theupper surface of the sample piece, 0.9% NaCl (−GSH) not containing GSHas a solvent or a 0.9% NaCl solution (+GSH) containing GSH at aconcentration of 1.0 mM as a solvent was used to seed about 1.0×10⁶cells/50 μL of E. coli. This bacterial suspension was covered with apolyethylene (PE) sheet so as not to dry the bacterial suspension. Then,the glass container was placed under static culture condition of 35±1°C. The PE sheet was removed 0 minute (immediately after seeding), 5minutes, and 10 minutes after seeding of E. coli, and 1 mL of 0.9%NaCl+0.1 mM EDTA-2Na was added to the bacterial suspension, and thebacterial suspension was collected by pipetting. A fluorescent dye wasadded to the collected bacterial suspension, and viability of E. coliwas determined by measuring fluorescence intensity. In order to avoid acontact between the back surface of the Cu sample piece and the solutionwhen collecting E. coli, the back surface of the Cu sample piece wascoated with a thin silicone sheet in advance.

As a control, as shown in FIG. 6(b), seeding of E. coli was performed ina similar manner to the above procedure except that a Cu sample piecewas not placed on the bottom of a glass container, and viability of E.coli after 10 minutes from seeding was measured.

Results are shown in FIG. 7 .

FIG. 7 shows the results of the viability of E. coli in the control (10minutes after seeding), the Cu sample (0 minute; immediately afterseeding), the Cu sample (5 minutes after seeding), and the Cu sample (10minutes after seeding). The bar graph on the left of each sample pieceshows the viability of E. coli when using the 0.9% NaCl (−GSH), and thebar graph on the right shows the viability of E. coli when using the0.9% NaCl solution (+GSH) to which GSH was added at a concentration of1.0 mM.

In the Cu sample immediately (0 minutes) after seeding of E. coli,viability of E. coli decreased due to the addition of GSH, but theviability was equivalent to the result of the control. On the otherhand, in the Cu samples of 5 minutes and 10 minutes after seeding of E.coli, viability decreased due to the addition of GSH, and the viabilitydecreased more than the result of the control. Particularly, thedecrease of the viability 10 minutes after seeding was considerable.

FIG. 8 shows results of viability of E. coli measured 5 minutes afterseeding of E. coli on a surface of a Cu sample piece in a manner similarto the above procedure. GSH was added at a concentration of 0 mM (noaddition), 1.0 mM, 2.0 mM, and 10.0 mM, and the results are shown inthis order from the left. In FIG. 8 , the longitudinal axis represents aratio of bacterial viability (relative viability) normalized to thebacterial viability in the control test as 1.

As in FIG. 7 , when a GSH concentration was 1.0 mM, bacterial viabilitydecreased more than a case without GSH (0.9% NaCl). However, when GSHwas added at a concentration of 2.0 mM or 10.0 mM, viability wasequivalent or greater than the result without GSH (0.9% NaCl).

These results show that improvement in antimicrobial activity of copperand a copper alloy by GSH (biological reducing substance) depends on theamount of Cu²⁺ eluted on the surface of the copper and the copper alloyand on physical and chemical states of the surface.

Example 6

In this Example, instead of Cu, CBRA was used as a copper alloy sample,and a simple test for antimicrobial activity using E. coli was performedin a similar manner to Example 5. Note that the time from seeding of E.coli to collection of a bacterial suspension was set to 5 minutes.

Results are shown in FIG. 9 .

In FIG. 9 , the bar graph on the left of each sample piece showsviability of E. coli when using 0.9% NaCl (−GSH), and the bar graph onthe right shows viability of E. coli when using a 0.9% NaCl solution(+GSH) to which GSH was added at a concentration of 1.0 mM. In FIG. 9 ,the longitudinal axis represents a ratio of bacterial viability(relative viability) normalized to the bacterial viability in thecontrol test as 1. FIG. 9 also shows the results using a Cu (C1020)sample piece for comparison.

In addition to Cu used in Example 5, the CBRA sample piece of thisExample reduced in bacterial viability compared with the control test.The results also show that addition of GSH further decreased theviability. These results show that both copper and a copper alloysignificantly improve in antimicrobial activity by GSH (biologicalreducing substance).

Example 7

In this Example, an antimicrobial activity test using E. coli based onthe antimicrobial activity test specified in JIS Z2801 was performed ina similar manner to Example 5 except that a 500-fold diluted nutrientbroth ( 1/500 NB) was used instead of 0.9% NaCl. Cu (C1020), CBRI, andC6932 were used as copper and copper alloy samples, and antimicrobialstainless steel (SS) and metallic silver (Ag) were used as samples forcomparison.

Results are shown in FIG. 10 .

In FIG. 10 , the bar graph on the left of each sample piece showsviability of E. coli when using 1/500 NB (−GSH), and the bar graph onthe right shows viability of E. coli when using a 1/500 NB solution(+GSH) to which GSH was added at a concentration of 1.0 mM. In FIG. 10 ,the longitudinal axis represents a ratio of bacterial viability(relative viability) normalized to the bacterial viability in thecontrol test as 1.

In each of the copper and copper alloy samples, the bacterial viabilitydecreased more than the result of the control test, and the viabilityfurther decreased by addition of GSH. On the other hand, in theantimicrobial SS and Ag, the bacterial viability did not decrease byaddition of GSH, and the viability did not decrease comparing to that inthe control test.

Example 8

In this Example, an antimicrobial activity test based on theantimicrobial activity test specified in JIS Z2801 was performed in asimilar manner to Example 7 except that S. aureus was used instead of E.coli. Cu (C1020), C6932, and C2680 were used as copper and copper alloysamples, and antimicrobial stainless steel (SS) and metallic silver (Ag)were used as samples for comparison.

Results are shown in FIG. 11 .

In FIG. 11 , the bar graph on the left of each sample piece shows theviability of S. aureus when using 1/500 NB (−GSH), and the bar graph onthe right shows the viability of S. aureus when using 1/500 NB (+GSH) towhich GSH was added at a concentration of 1.0 mM. In FIG. 11 , thelongitudinal axis represents a ratio of viability (relative viability)normalized to the viability in the control test as 1.

In each of the copper and copper alloy samples, the bacterial viabilitydecreased comparing to that in the control test, and the viabilityfurther decreased by addition of GSH. On the other hand, in theantimicrobial SS and Ag, the bacterial viability did not decrease byaddition of GSH nor comparing to that in the control test.

Example 9

In this Example, antimicrobial activity tests using E. coli and S.aureus based on the antimicrobial activity test specified in JIS Z2801were performed in a similar manner to Example 7 except that copper andcopper alloy samples were exposed to the atmosphere for a certain periodof time. Cu, CBRA, and C2680 were used as copper and copper alloysamples.

Results are shown in FIGS. 12(a) and 12(b).

In FIGS. 12(a) and 12(b), the bar graph on the left of each sample pieceshows the viability of E. coli (FIG. 12(a)) or S. aureus (FIG. 12(b))when using 1/500 NB NaCl (−GSH), and the bar graph on the right showsthe viability of E. coli or S. aureus when using 1/500 NB (+GSH) towhich GSH was added at a concentration of 1.0 mM. In FIGS. 12(a) and12(b), the longitudinal axis represents a ratio of viability (relativeviability) normalized to the bacterial viability in the control test as1.

In each of the copper and copper alloy samples, the viability of E. coliand S. aureus decreased comparing to those in the control tests, and theviability further decreased by addition of GSH.

Although it is difficult to simply compare the results in this Examplewith the results in Example 7 (FIG. 10 ) and Example 8 (FIG. 11 )because types (compositions) of the copper alloy samples are different,the effects on the exposed materials caused by addition of GSH wereprominent in this Example. These results indicate that the effects of areducing agent present on a surface of copper and a copper alloy and theeffects of copper (I) act additively and synergistically so as topromote generation of reactive oxygen species, which accelerates themanifest of antimicrobial actions on the surface of the copper and thecopper alloy and enhances their antimicrobial activity.

With regard to the exposed materials of Cu, CBRA, and C2680 used in thisExample, surfaces of the samples were exposed in the atmosphere forthree months, and the surfaces were analyzed by XPS. The analysis showsthat the proportions of Cu₂O in Cu, CBRA, and C2680 were 83.0%, 90.0%,and 92.9%, respectively. For comparison, a proportion of Cu₂O on anexposed CBRI surface was measured under similar conditions, and theresult was 84.6%. As described above, the surface of exposed materialscontains Cu₂O in a higher proportion than CuO, which indicates thepresence of copper (I) in addition to copper (II) ions has significantcontributions. This indicates that, in addition to the effects of abiological reducing substance, the presence of copper (I) on a surfaceof copper or a copper alloy may be a promising factor for improvingantimicrobial activity of the copper or the copper alloy. This is one ofanalytical data that supports the findings of the present inventors.

The embodiments of the present invention have been described in detail.However, specific embodiments are not limited to these embodiments, andany modification in design within the gist of the present is included inthe present invention.

The invention claimed is:
 1. A surface treatment method for articles,equipment, components, indoor floors, or wall surfaces, in which copperor a copper alloy is used in whole or in part, the method comprising:preparing a reducing agent solution containing a biological reducingsubstance, the biological reducing substance being at least one selectedfrom the group consisting of reduced glutathione, N-acetylcysteine,sodium ascorbate, sodium sulfite, and cysteine, and treating the surfaceof the articles, the equipment, the components, the indoor floors, orthe wall surfaces with the reducing agent solution.
 2. The surfacetreatment method according to claim 1, wherein the copper or the copperalloy is a wrought copper and copper alloy product.
 3. The surfacetreatment method according to claim 1, wherein the treating is performedon the surface of the article, in which copper or a copper alloy is usedin whole or in part, the article is a porous body, and the copper or thecopper alloy is a fiber, a particle, or foil of copper or a copper alloycontained in the porous body.
 4. The surface treatment method accordingto claim 3, wherein the porous body is a woven fabric, a non-wovenfabric, or a sponge.
 5. The surface treatment method according to claim1, wherein the treating is performed at a relative humidity of 70% RH orless.
 6. The surface treatment method according to claim 1, wherein thecopper or the copper alloy is a Cu—Zn alloy, a Cu—Ni—Zn alloy, aCu—Sn—Ni—Zn alloy, or a Cu—Si—Pb—P—Zn alloy.
 7. The surface treatmentmethod according to claim 1, wherein the biological reducing substanceis reduced glutathione, and the reduced glutathione in the reducingagent solution has a concentration of 0.5 to 2.0 mM.
 8. The surfacetreatment method according to claim 1, wherein the copper or the copperalloy includes an oxide layer, and the oxide layer contains 80.0% ormore of Cu₂O and 20.0% or less of CuO in an area from the surface of theoxide layer to 1 μm measured by X-ray photoelectron spectroscopy.
 9. Asterilization method comprising: conducting the surface treatment methodaccording to claim 3, and thereafter bringing the article into contactwith the surface of an object to sterilize the surface of the object.10. The sterilization method according to claim 9, wherein the porousbody is a woven fabric, a non-woven fabric, or a sponge.